Myosin is responsible for a variety of contractile processes including contraction of cardiac muscle. Myosin consists of three different subunits, the myosin heavy chain (MHC), and two different light chains, MLC1 and MLC2. Because mutations in any of these subunits have been shown to produce cells with similar phenotypes, it seems likely that mutations in any of the cardiac myosin subunits could result in defects in cardiac contractility. Recently mutations in the cardiac beta-MHC have been shown to produce about 50% of the cases of familial hypertrophic cardiomyopathy. The basis for the remaining cases are thought to be mutations in gene other than the MHC. In this application we propose a comprehensive series of experiments to employ rat ES cells that we have recently generated, as hosts for the molecular genetic manipulation of cardiac MLC gene expression. These manipulations involve the use of gene targeting to disrupt the cardiac MLC isoforms and the use of expression vectors to express cardiac MLC isoforms in both wildtype and mutant rat ES cells. We will examine the resulting cells to determine the consequences of these molecular genetic manipulations. The mutant cells will be employed to generate chimeric rats and the consequences of MLC manipulations on the development and function of the heart will be investigated. Four specific aims are proposed: (1) to further characterize our rat ES cells, (a) establishing their competence to contribute to the germ line of chimeric rats, (b) to establish additional rat ES cell lines, and (c) to optimize rat chimera production using these cells; (2) to employ gene replacement to create rat ES cells which no longer express the ventricular or atrial MLCs and analyze the consequences for the contractile properties of these mutant cell lines; (3) to employ the MLC mutant ES cells as hosts for the expression of wildtype and mutant MLC isoforms to investigate the contribution of MLC isoforms to myosin function; and (4) to use these cell lines to produce rats displaying altered expression of cardiac MLC isoforms. These animals will be used to study the developmental and physiological consequences of myosin mutations. These studies may produce rat models for cardiomyopathies or other myosin-based pathologies. Once established, these animal models may be useful for the development of therapeutic modalities and interventions which might be effective in treating cardiovascular diseases in humans.